ESTROGEN RECEPTOR Beta PARTIAL AGONIST HAVING ESTROGEN RECEPTOR Alpha INHIBITORY EFFECT, AND GYNECOLOGICAL DISEASE THERAPEUTIC AGENT USING SAME

ABSTRACT

[Problem] To provide a therapeutic agent for estrogen-dependent gynecological diseases such as endometriosis, uterine fibroids, and uterus adenomyosis. [Solution] To use an estrogen receptor α-inhibiting β partial agonist represented by the formula (a), a pharmaceutically-acceptable salt thereof, or a hydrate of either of the afore-mentioned, as an active ingredient.

FIELD OF THE INVENTION

The present invention relates to an estrogen receptor β partial agonisthaving estrogen receptor α inhibitory effect, and a gynecologicaldisease therapeutic agent using the same.

BACKGROUND OF THE INVENTION

There are diseases caused by abnormality of estrogen or progesterone asorganic gynecological diseases. Examples of the diseases includeendometriosis, uterine fibroids, and uterus adenomyosis.

As treatment of such diseases, administration of gonadotropin agonist(GnRH agonist), as a drug therapy, is known, however, a period for drugadministration is restricted because of the side effects such as bonemineral loss and ovarian dysfunction symptoms, thus no gynecologicaldisease therapeutic agents for long-term use have existed yet, and sosurgical therapy such as surgical resection is still a first choice.

Therefore, for the treatment of gynecological disease, a drug effectivefor long-term administration to inhibit the growth of ectopicendometrial tissue and suppress or improve the intra-uterine fibroidsand uterus adenomyosis has been desired. It has also been desired torelief pains such as lower abdominal pain and low back pain caused bythese gynecological diseases.

Conventionally, as a drug to inhibit binding of estrogen to the receptorantagonist, tamoxifen (Registered Trademark: Nolvadex) with anindication of breast cancer has been used since the 1980s in Japan.However, it is reported that this drug causes uterine body cancer,uterine sarcoma or endometriosis, thus it may not be used to treatestrogen-dependent diseases including endometriosis. The mechanism ofproliferative effects of the drug against the endometrium is caused byits endogenous partial agonistic action to the estrogen receptor.

Then, fulvestrant (Registered Trademark: Faslodex) was approved as atherapeutic agent for postmenopausal breast cancer. This drug, unliketamoxifen, does not provide endogenous partial agonist action to theestrogen receptor. However, this drug has low gastrointestinalabsorption rate as well as short half-life even when intravenouslyadministrating, thus a high capacity intramuscularly administration isnecessary, and thus the dosage form is intramuscular injection.Therefore, it will never be used orally for treating estrogen-dependentdiseases.

Further, as a selective estrogen receptor modulator (hereinafter,sometimes abbreviated as “SERM”), raloxifene (Registered Trademark:Evista) has been approved with an indication for osteoporosis treatment.However, this drug is an osteoporosis therapeutic agent having a bonemass-increasing by estrogen receptor partial agonist activity, thus itwill never be used for treating estrogen-dependent diseases, that isgynecological diseases caused by ectopic endometrial tissue growth suchas endometriosis, uterine fibroids, and uterus adenomyosis.

Other selective estrogen receptor modulators, (7α)-21-[4-[(diethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-triene-3-ol ora pharmaceutically acceptable salt thereof, which have been developed asa breast cancer therapeutic agent, are known (see Patent documents 1 and2).

PRIOR ART DOCUMENTS Patent Documents

-   Patent document 1: WO 2001/058919-   Patent document 2: WO 1999/033859

Non-Patent Documents

-   Non-patent document 1: Clinical Cancer Research, vol. 11, 315-322,    2005-   Non-patent document 2: Metabolism and Disposition, vol. 34 (2),    331-338, 2006-   Non-patent document 3: BBRC, vol. 312, 656-662, 2003-   Non-patent document 4: Journal of Clinical Pharmacology and    Therapeutics, vol. 30, 456-470, 2005-   Non-patent document 5: Clinical Cancer Research, vol. 10, 5425-5431,    2004-   Non-patent document 6: Basic & Clinical Pharmacology & Toxicology,    vol. 104, 352-359, 2009-   Non-patent document 7: Annals of Oncology, vol. 714, 1-6, 2009

SUMMARY OF THE INVENTION Problem to be Solved by Invention

The objective of the present invention is to provide a therapeutic agentof estrogen-dependent gynecological diseases such as endometriosis,uterine fibroids, and uterus adenomyosis.

Means for Solving the Problem

As a result of intensive studies on the above problems, quiteunexpectedly, the present inventors have found that the compoundrepresented by the following formula (a) synthesized and clinicallystudied to treat breast cancers worked as an estrogen receptorα-inhibiting β partial agonist and showed an excellent therapeuticeffect on estrogen-dependent gynecological diseases, and accomplishedthe present invention.

The present invention provides a specific embodiment described in thefollowing (1) to (3).

(1) An estrogen receptor α-inhibiting β partial agonist represented bythe following formula (a) represented by the following structuralformula (a).

(2) A gynecological disease therapeutic agent comprising the estrogenreceptor α inhibiting β partial agonist of the (1) above, apharmaceutically acceptable salt thereof, or a hydrate of either of theafore-mentioned as an active ingredient. (3) The gynecological diseasetherapeutic agent of the (2) above, wherein the gynecological diseasecomprises endometriosis, uterine fibroids, and/or uterus adenomyosis.

Further, the present invention provides a specific embodiment describedin the following (4) to (8).

(4) A method for treating gynecological diseases; a method for treatingendometriosis, uterine fibroids, and/or uterus adenomyosis; and a methodfor treating endometriosis, uterine fibroids, and/or uterus adenomyosisin premenopausal patients; comprising a step of administering theestrogen receptor α-inhibiting β partial agonist of the (1) above, apharmaceutically acceptable salt thereof, or a hydrate of either of theafore-mentioned, as an active ingredient.(5) A method for alleviating gynecological diseases; a method foralleviating endometriosis, uterine fibroids, and/or uterus adenomyosis;and a method for alleviating endometriosis, uterine fibroids, and/oruterus adenomyosis in premenopausal patients; comprising a step ofadministering the estrogen receptor α-inhibiting β partial agonist ofthe (1) above, a pharmaceutically acceptable salt thereof, or a hydrateof either of the afore-mentioned, as an active ingredient.(6) A use of the estrogen receptor α-inhibiting β partial agonist of the(1) above, a pharmaceutically acceptable salt thereof, or a hydrate ofeither of the afore-mentioned for treating gynecological diseases; fortreating endometriosis, uterine fibroids, and/or uterus adenomyosis; andfor treating endometriosis, uterine fibroids, and/or uterus adenomyosisin premenopausal patients.(7) A use of the estrogen receptor α-inhibiting β partial agonist of the(1) above, a pharmaceutically acceptable salt thereof, or a hydrate ofeither of the afore-mentioned for manufacturing therapeutic agents fortreating gynecological diseases; for manufacturing therapeutic agentsfor treating endometriosis, uterine fibroids, and/or uterus adenomyosis;and for manufacturing therapeutic agents for treating endometriosis,uterine fibroids, and/or uterus adenomyosis in premenopausal patients.(8) Oral formulations comprising the estrogen receptor α-inhibiting βpartial agonist of the (1) or (2) above as an active ingredient.

Effect of the Invention

According to the present invention, a drug for treatingestrogen-dependent gynecological diseases such as endometriosis, uterinefibroids, and uterus adenomyosis may be provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A photograph showing grafted uterine strip of the ratendometriosis model in the examples.

FIG. 2: A graph showing anti-angiogenic related PEDF gene expressionpromoting effect of the endometrial implants in the examples.

FIG. 3: A graph showing inflammation-associated IL-6 gene expressioninhibitory effect of the endometrial implants in the examples.

DESCRIPTION OF THE EMBODIMENTS

Hereinafter will be described in detail embodiments of the presentinvention, the following embodiments are examples for explaining thepresent invention, the present invention is not limited thereto, it canbe practiced with various modifications without departing from the scopeof the subject matter. In the present description, for example,reference numerical range of “1 to 100” is intended to include both ofthe upper limit value “100” and the lower limit value “1”. In addition,the other numerical ranges are referred in the same manner.

(Estrogen Receptor α Inhibitors, Estrogen Receptor β Partial Agonists)

A compound represented by the following formula (a) used in the presentinvention is orally active, and has an estrogen receptor α inhibitoryeffect and an estrogen receptor β partial agonist action.

A compound represented by the above formula (a) used in the presentinvention may be used in free form, in pharmaceutically acceptable saltform, or in hydrate form. The pharmaceutically acceptable salts include,but are not limited to, inorganic salts such as hydrochloride, sulfate,nitrate, hydrobromide and phosphate; and organic salts such as acetate,trifluoroacetate, lactate, propionate, tartrate, glycolate, pyruvate,oxalate, malate, malonate, succinate, maleate, fumarate, tartrate,citrate, benzoate, cinnamate, mandelate, methanesulfonate,ethanesulfonate, p-toluenesulfonate and salicylic acid; among thesesalts, the organic salts are preferred, and citrate, fumarate,succinate, benzoate and malonic acid are more preferred, and citrate isyet more preferred (hereinafter, citrate of the compound of the presentinvention may be abbreviated to “SR16234”).

The compound represented by the above formula (a) used in the presentinvention, a pharmaceutically acceptable salt thereof, and a hydratethereof may be prepared, for example, according to the methods describedin the Patent Documents 1 and 2.

The compound represented by the above formula (a), a pharmaceuticallyacceptable salt thereof, and a hydrate thereof, as shown in the examplesbelow, has an anti-proliferative activity in ectopic endometrial tissue,which is stable for a long period of time. Further, those compounds canbe administered orally with a low toxicity, and may be safely used evenby premenopausal patients. Thus, the drugs of the present invention areespecially useful as therapeutic agents for estrogen-dependentgynecological diseases such as endometriosis, uterine fibroids, anduterus adenomyosis.

(Gynecological Disease Therapeutic Agents)

The gynecological disease therapeutic agent of the present inventionincludes at least the compound represented by the formula (a), apharmaceutically acceptable salt thereof, or a hydrate of either of theafore-mentioned, as an active ingredient. The gynecological therapeuticdrug of the present invention may include other components as needed.Examples of the other components include, but are not limited to,pharmaceutical additives such as stabilizers, surfactants, plasticizers,lubricants, solubilizers, buffering agents, sweetening agents,substrates, adsorbents, taste masking agents, binders, suspendingagents, antioxidants, brightening agents, coating agents, flavoringagents, fragrances, humectants, wetting modifiers, defoamers, chews,fresheners, coloring agents, sugar coating agents, isotonic agents, pHadjusting agents, softeners, emulsifiers, adhesives, adhesion enhancers,thickeners, thickening agents, foaming agents, excipients, dispersingagents, propellants, disintegrating agents, disintegrating aids,aromatics, desiccants, antiseptic agents, preservatives, soothingagents, solvents, dissolving agents, solubilizing agents and fluidizingagents.

A dosage form of the gynecological disease therapeutic agents of thepresent invention may be selected, but is not limited to, depending onthe patients age, weight, diseases, symptoms or the degree of thediseases or symptoms. Examples of the dosage form include oraladministration in tablets (including sublingual tablets and orallydisintegrating tablets), granules, powders, solutions, syrups (includingdry syrups.), jelly and capsules (including soft capsules andmicrocapsules); and parental administration in injections (such assubcutaneous injections, intravenous injections, intramuscularinjections and intraperitoneal injections), suppositories (includingrectal suppositories and vaginal suppositories), inhalations,percutaneous formulations, eye drops and nasal drops. These may becontrolled release formulation such as rapid-release formulation orsustained-release formulation. Among these formulations, oraladministration in oral formulations is preferable from the view of easyto apply and medication compliance, and easy to reduce cost. As oralformulations, tablets, solutions and syrups are preferred, and tabletsare more preferred.

A dosage amount of gynecological disease therapeutic agents of thepresent invention may be decided depending on the patients age, weight,diseases, symptoms or the degree of the diseases or symptoms. It is notparticularly limited as long as pharmaceutically effective amount, butit may be, for oral formulation, in the range of 0.05 to 20 mg/kg bodyweight in free form in terms of the compound represented by the aboveformula (a), more preferably in the range of 0.06 to 18 mg/kg bodyweight, yet more preferably in the range of 0.07 to 17 mg/kg bodyweight, it may be preferable, for parental formulation, in the range of0.001 to 10 mg/kg body weight, more preferably in the range of 0.002 to9 mg/kg body weight, yet more preferably in the range of 0.003 to 8mg/kg body weight.

The gynecological disease therapeutic agent of the present invention maybe administered once or divided into several times. On administration,the dosage amount per day may be usually 0.1 to 5000 mg/kg, and a singledose or divided doses is desirable.

In a formulation of the gynecological disease therapeutic agent of thepresent invention, additives known in the art may be used, and methodsknown in the art, for example, methods described in the sixteenthJapanese Pharmacopoeia may be applied. For example, when preparing anoral solid formulation, to the active ingredient, excipients, binders,disintegrators, lubricants, coloring agents, taste masking agents orflavoring agents were added, followed by molded, granulated andencapsulated according to a conventional method to produce coatedtablets, granules, powders, capsules, and the like. When preparing anoral liquid preparation, to the active ingredient, solvents such aspurified water or ethanol, solubilizing agents, suspending agents,isotonic agents, taste masking agents, buffering agents, stabilizingagents or flavoring agents were added, followed by prepared and packedaccording to a conventional method to produce oral solutions, syrups,and the like. When preparing injections, to the active ingredient, pHadjusting agents, buffering agents, stabilizers, tonicity agents, orlocal anesthetics were added, followed by aseptically encapsulated intoa container according to a conventional method to prepare subcutaneous,intramuscular, intravenous injections, and the like. When preparingrectal suppositories, to the active ingredient, excipients orsurfactants were added, followed by mixed and molded according to aconventional method. When preparing formulations such as ointments,pastes, creams and gels, to the active ingredient, base materials suchas white petrolatum and paraffin, stabilizers, wetting agents, orpreservatives such as methyl parahydroxybenzoate were added, followed bymixed according to a conventional method. When preparing adhesivepatches, to the support substrates such as woven fabrics, nonwovenfabrics, or plastic films, the ointments, creams, gels, pastes or thelike may be applied by a conventional method.

The gynecological disease therapeutic agent of the present invention istargeted to estrogen-dependent gynecological diseases. As describedabove, since having anti-proliferative activity in ectopic endometrialtissues, targeting to endometriosis, uterine fibroids and uterusadenomyosis is preferred, and the agent may be safely used bypremenopausal patients due to low toxicity. The agent may also alleviateor treat menorrhagia, irregular vaginal bleeding, dysmenorrhea, andpressure symptoms caused by these diseases.

EXAMPLES

Hereinafter, the present invention will be described in more detail byreference to the following various test results, but the inventionshould not be construed as being limited thereto.

Test Example 1: Confirmation of Endometriosis Inhibiting Activity<Preparation of Endometriosis Model> [1] Ovariectomy 1. AnalgesicTreatment

Buprenorphine hydrochloride (Lepetan injection 0.3 mg, OtsukaPharmaceutical Co., Ltd., 0.2 mg/mL of which was diluted to 6.7 timeswith a physiological saline solution to provide 0.03 mg/mL solution whenusing) was administered subcutaneously into the back of the rats at adose of 0.0024 mg/body (0.08 mL/body). The administration period was twodays, which includes twice before and after the model preparation on theday when the model was prepared, and twice per day on the next day. Itshould be noted that the dosage amount of 0.0024 mg/body was the onethat satisfied a recommended dose of 0.01 mg/kg in 250 g-weight rat(0.0096 mg/kg) (estimated weight upon model preparation: 180 to 240 g).

2. Ovariectomy method

Under 2% inhalation anesthesia, left and right flanks of the rats(operation site) were shaved, and the skin of one of the flasks wasdissected. After peeling off the skin, the muscle layer was cut to openusing a retractor so as to protrude it to the outside of the body withfat tissue around the ovary, then the fallopian tubes were ligated witha silk thread (sterile suture) followed by removing the ovary includingthe fat tissue. The peeled muscle layer and one part of the peritoneumwere sutured with silk thread (sterile suture), then kanamycin solution,antibiotics, (manufactured by Meiji Seika Pharma Co., Ltd., “Meiji,”kanamycin sulfate injection of 1000 mg was diluted to 5 mg/mL withinjection solvent) was applied to the operation site. The dissected skinwas sutured with silk thread (sterile suture). Another ovary in theopposite side was removed in the same manner. At the time of theoperation, sterile gloves were used, and surgical instruments used weresterilized by an autoclave and beads sterilizer. The surgical area wasshaved with an electric clipper followed by disinfected with isodinealcohol and covered with a sterile bedsheet and a covering cloth.

3. E2 Treatment

Since the day of ovariectomy, after it was performed, E2 (β-Estradiol, 3μg/mL animal) was subcutaneously administered to the back of the ratsevery day for 2 weeks. E2 (3 μg/mL animal) was administered in the samemanner during 4 weeks after model preparation on the date that the modelwas prepared.

[2] Grouping and Model Preparation 1. Grouping

Thirteen days after ovariectomy (day 0 counting), the rats were dividedinto four groups (N=8) as the total weight on the grouping day wasuniform in each group. Animals excluded from the groups were distributedto the Sham group.

2. Method of Model Preparation

On the next day of the grouping, models were prepared in accordance withUchiide et al method, which was partially modified Vernon et al method.Rats were placed under inhalation anesthesia of 2% isoflurane, and madean incision in the abdomen along the midline, then the right uterinehorn was excised and cut in half along the vertical line after removingthe fat cells around the excised uterine horn. The uterine horn was madean incision along the horizontal axis to cut out two strips ofapproximately 5 mm×5 mm. The serosal surface of the abdominal cavity andthe endometrial surface of the uterus strips were faced and sawed up thefour corners thereof with a bioabsorbable suture (6-0, PDS-II,manufactured by Ethicon) to graft the uterine horns into the right andleft abdominal walls one by one. In order to avoid adhesion, theabdominal cavity was washed with saline (manufactured by OtsukaPharmaceutical Factory). Then, the abdominal wall fascia was sawed upwith a bioabsorbable suture (4-0, monocryl, manufactured by Ethicon) andthe skin was sawed up with a silk suture (4-0, blade silk, manufacturedby Ethicon).

[3] Sham Tissue Collection and Wet Weight Measurement 1. Dissection

On the previous day of the model preparation (the grouping date), underinhalation anesthesia of 2% isoflurane, an incision was made in theabdomen of the rats. Subsequently, about 5 mL of blood was collectedwith a syringe from the abdominal vena cava and collected in a VenojectII vacuum blood collection tube (EDTA-2K), then cut the abdominal venacava and abdominal aorta to euthanize by exsanguination. The rightuterine horn was excised and cut out two strips (approximately 5 mm×5mm), then cut out strips (approximately 5 mm×5 mm) from the graft sites,the right and left abdominal walls (muscle layer and peritoneum). Afterthat, wet weight of each strip and abdominal wall was measured. Thenpituitary, adrenal glands (both sides), left uterine horn, mammary gland(excised with the nipples, three on the lower abdomen, a sample for RNAmeasurement was approximately 3 mm in diameter) and femur (left side)were excised.

2. EDTA Added Blood Treatment

The collected EDTA-added blood was centrifuged for 10 minutes at 4° C.at 1800×g to give plasma (2 mL or more). The obtained plasma was frozenand stored in an ultra-low temperature freezer.

3. Weight of Excised Organs

As for the pituitary, adrenal glands (both sides), left uterine horn andfemur, wet weight was measured. The femur bone after wet weightmeasurement was discarded.

4. Treatment of Excised Organs

The pituitary, adrenal gland (right), uterine horn strip (approximately5 mm mm), graft site abdominal walls (muscle layer and peritoneum,approximately 5 mm×5 mm) (one each), left uterine horn (portioncollected from the above organs), and mammary glands (approximately 3 mmin diameter) were immersed in the RNA later solution and stored in arefrigerator for 1 day, then the RNA later solution was removed andstored in a very low temperature freezer (sample for RNA measurement).The remainder of the adrenal gland (right) was discarded, the remainderof the adrenal gland (left), mammary gland (one) and the remainder ofeach organ was frozen in liquid nitrogen.

5. Pathological Specimen

The mammary gland, the remaining uterine horn strip (approximately 5mm×5 mm) and the graft site abdominal walls (muscle layer andperitoneum, approximately 5 mm×5 mm) (one each) were immersed in 10%neutral buffered formalin solution and fixed, respectively, thenparaffin block-embedded specimens were prepared.

<Preparation and Administration of the Drug> [1] Preparation Method(Undiluted Solution)

A required amount of SR16234 was weighed, pulverized with an agatemortar and pestle, and suspended in 0.5% carboxymethylcellulose sodium(CMC-Na) solution to prepare a 2 mg/mL solution, then used as undilutedsolution. Preparation was performed using a volumetric flask. Theobtained undiluted solution was stored in a cold place, in arefrigerator at the test substance storage room J009 (acceptabletemperature range: 1 to 15° C.) after the preparation, then used within7 days after the date of preparation. The remaining undiluted solutionwas discarded.

[2] Preparation Method (Solution for Administration)

The undiluted solution (2 mg/mL solution) was serially diluted with 0.5%CMC-Na aqueous solution to prepare 0.2, 0.06 and 0.02 mg/mL solution wasrespectively prepared. As a solution for administration for the vehiclecontrol group, 0.5% CMC-Na aqueous solution was used. Preparation wascarried out using a graduated cylinder or volumetric flask. Theremaining undiluted solution was discarded.

[3] Drug Administration

The administration was made orally, and the drug was forciblyadministered into a stomach once a day using a flexible oral sonde andsyringe for 28 consecutive days.

<Evaluation of Endometriosis Inhibiting Activity> [1] Dissection

On the next day of the final administration, the abdominal dissectionwas made under inhalation anesthesia of 2% isoflurane, and the grafteduterine strips (two pieces) were excised, and photographed with adigital camera. Subsequently, about 5 mL of blood was collected with asyringe from the abdominal vena cava and collected in a Venoject IIvacuum blood collection tube (EDTA-2K), then cut the abdominal vena cavaand abdominal aorta to euthanize by exsanguination. Then pituitary,adrenal glands (both sides), graft uterine strips (two pieces), leftuterine horn, mammary gland (excised with the nipples, three on thelower abdomen, a sample for RNA measurement was approximately 3 mm indiameter) and femur (left side) were excised.

[2] Photography

The grafted uterine strips were photographed with a digital camera oneby one along with a ruler.

[3] EDTA Added Blood Treatment

The collected EDTA-added blood was centrifuged for 10 minutes at 4° C.at 1800×g to give plasma (2 mL or more). The obtained plasma was frozenand stored in an ultra-low temperature freezer.

[4] Weight of the Excised Organs

As for the pituitary, adrenal glands (both sides), grafted uterine strip(one piece), left uterine horn and femur, wet weight was measured. Thefemur bone after wet weight measurement was discarded.

[5] Treatment of the Excised Organs

The pituitary, adrenal glands (right), graft uterine horn (one piece),left uterine horn (portion collected from the above organs), and mammarygland (approximately 3 mm in diameter) were immersed in the RNA latersolution and stored in a refrigerator for 1 day, then the RNA latersolution was removed and stored in a very low temperature freezer(sample for RNA measurement). The remainder of the adrenal gland (right)was discarded, the adrenal gland (left), mammary gland (one) and theremainder of each organ were frozen in liquid nitrogen.

[6] Pathological Specimen

The mammary gland and the rest of the grafted uterine strip (one piece)were immersion-fixed in 10% neutral buffered formalin solution toprepare a paraffin block-embedded specimen.

[7] Data Analysis 1. Calculation of Wet Weight of the Grafted UterineStrips

The wet weight of each grafted uterine strip (left and right) wascalculated by subtracting the average value of the wet weight of theleft and right abdominal walls in the Sham group.

2. Calculation of Suppression Rate (%) of the Grafted Uterine StripWeight

The suppression rate of the grafted uterine strip weight was calculatedon the basis of the total average wet weight of the left and grafteduterine strips. Here, using the average wet weight of the grafteduterine strip in the vehicle control group as a basis, the suppressionrate (%) of the grafted uterine strip weight in the vehicle controlgroup and test sample group was calculated based on the followingequation. The calculation results are shown in Table 1. Suppression rateof grafted uterine strip weight (%)=(average wet weight of the grafteduterine strip in the vehicle control group−wet weight of the grafteduterine strip of each individual)×100/(average wet weight of the grafteduterine strip in the vehicle control group)

TABLE 1 Group Suppression rate (% ± SE) Vehicle control group  0.0 ±19.0 SR16234 0.1 mg/kg p.o. 36.4 ± 25.2 SR16234 0.3 mg/kg p.o. 63.2 ±17.7 SR16234 1.0 mg/kg p.o. 97.7 ± 21.2

3. Suppression Rate of the Grafted Uterine Strip Weight

The suppression rate of the grafted uterine strip in the vehicle controlgroup after administering 0.5 w/v % of sodium carboxymethyl celluloseaqueous solution (0.5% CMC-Na aqueous solution) was set as 0% tocalculate the suppression rate of the grafted uterine strip (averagevalue) in the 0.1, 0.3 and 1.0 mg/kg SR16234 administered groups, sothat it was 26%, 45%, and 68%, respectively.

TABLE 2 Group Suppression rate (%) Vehicle control group 0.0 SR16234 0.1mg/kg p.o. 26.0 SR16234 0.3 mg/kg p.o. 45.0 SR16234 1.0 mg/kg p.o. 69.0

As shown in Table 2, the increase suppression rate of the grafteduterine strip weight of SR16234 administered group was increased withincreasing dose. Especially, the 1.0 mg/kg administered group showed aparticularly significant suppression rate of the grafted uterine stripweight compared to the vehicle control group. From the observationabove, it was confirmed an improving effect on endometriosis by thegynecological diseases therapeutic agent of the present invention.

Test Example 2: Confirmation of Endometriosis Inhibiting Activity in theEndometriosis Model <Total Wet Weight of the Grafted Uterine Strip andthe Abdominal Wall>

A total wet weight of the left and right uterine strips and abdominalwall in the Sham group was 0.2596 g. In addition, a total wet weight ofthe left and right grafted uterine strips (including the abdominal wall)in the vehicle control group and 0.1, 0.3 and 1.0 mg/kg SR16234administered groups was respectively 0.4785 g, 0.3988 g, 0.3401 g and0.2647 g.

After the ovariectomy, 3 μg/mL/animal of E2 (β-Estradiol) wassubcutaneously administered to the rats every day to maintain hormonebalance, then using the rats, it was examined the improving effect byadministration of SR16234 to “the endometriosis model” with transplantedthe right uterine horn to the left and right abdominal walls.Administration of SR16234 was made orally once per day with three dosesof 0.1, 0.3 and 1.0 mg/kg for consecutive 28 days from the day when theendometriosis model was prepared.

TABLE 3 Total wet weight of the Group grafted uterine strips (g) Vehiclecontrol group 0.4785 SR16234 0.1 mg/kg p.o. 0.3988 SR16234 0.3 mg/kgp.o. 0.3401 SR16234 1.0 mg/kg p.o. 0.2647

As shown in Table 3, the total wet weight of the grafted uterine strips(including abdominal wall) in the SR16234 administered group isdecreased with increasing dose. Especially, 1.0 mg/kg SR16234administered group shows a particularly significant suppression ratio ascompared to the vehicle control group. From the above, it was confirmedthe improving effect by administration of the gynecological diseasetherapeutic agents of the present invention on the ovariectomized ratendometriosis model.

Test Example 3: Confirmation of Activities to the Bone Weight, UterineWeight, and Pituitary Weight <Wet Weight of Each Organ> [1] Sham Group

The wet weight of pituitary, adrenal gland (left), adrenal gland(right), adrenal glands (the sum of the left and right), left uterinehorn and femur (left) in the Sham group was 0.0186 g, 0.0353 g, 0.0296g, 0.0649 g, 0.1751 g and 0.7083 g, respectively.

[2] Vehicle Control Group

The wet weight of pituitary, adrenal gland (left), adrenal gland(right), adrenal glands (the sum of the left and right), left uterinehorn and femur (left) in the vehicle control group was 0.0271 g, 0.0468g, 0.0425 g, 0.0893 g, 0.1428 g and 0.7589 g, respectively.

[3] 0.1 mg/kg SR16234 Administered Group

The wet weight of pituitary, adrenal gland (left), adrenal gland(right), adrenal glands (the sum of the left and right), left uterinehorn and femur (left) in the 0.1 mg/kg SR16234 administered group was0.0226 g, 0.0438 g, 0.0400 g, 0.0838 g, 0.1261 g and 0.7597 g,respectively.

[4] 0.3 mg/kg SR16234 Administered Group

The wet weight of pituitary, adrenal gland (left), adrenal gland(right), adrenal glands (the sum of the left and right), left uterinehorn and femur (left) in the 0.3 mg/kg SR16234 administered group was0.0187 g, 0.0398 g, 0.0381 g, 0.0779 g, 0.1119 g and 0.7598 g,respectively.

[5] 1.0 mg/kg SR16234 Administered Group

The wet weight of pituitary, adrenal gland (left), adrenal gland(right), adrenal glands (the sum of the left and right), left uterinehorn and femur (left) in the 1.0 mg/kg SR16234 administered group was0.0174 g, 0.0375 g, 0.0357 g, 0.0732 g, 0.1016 g and 0.7301 g,respectively.

[6] Results

It was confirmed that the SR16234 administered groups exhibited adecreasing trend associated with the dose increase in comparison withthe vehicle control group. It was also confirmed that 0.3 mg/kg and 0.1mg/kg SR16234 administered group exhibited a very mild weightsuppression trend in comparison with the vehicle control group after theDay 14.

Test Example 4: Effects on Gene Expression of the Endometriosis Model<Gene Expression Analysis of Endometrial Implants> [1] Summary

Total RNA was extracted from 8 target genes of the rat-derivedendometrial graft (27 specimens), then quality of the RNA was inspectedwith a spectrophotometer and bio-analyzer, and then reversetranscription reaction of the RNA was performed, followed by geneexpression analysis was performed according to TaqMan method.

1. Main Instruments Used in the Test

-   -   NanoDrop 1000 (manufactured by Thermo Fisher Scientific)    -   Agilent 2100 Bioanalyzer (manufactured by Agilent)    -   Step One Plus Real-Time PCR System (manufactured by Life        Technologies)

2. Main Reagents and Apparatus Used in the Test <RNA Extraction Reagent>

RNeasy Fibrous Tissue Mini Kit (manufactured by QIAGEN)

<Reverse Transcriptase>

SuperScript VILO Master Mix (manufactured by Life Technologies)

<TaqMan Reagent>

TaqMan Gene Express Assay (manufactured by Life Technologies)

TABLE 4 Index Gene name TaqMan Assay ID 1 Era Rn01640372_ml 2 ErbRn00562610_ml 3 PR-A Rn01448227_ml 4 PR-B Rn01448227_ml 5 AromataseRn01422546_ml 6 VEGF Rn01511601_ml 7 IL-6 Rn01410330_ml 8 MCP-1Rn00580555_ml 9 PEDF Rn00709999_ml — GAPDH Rn01775763_ml

[2] Methods for Conducting the Test 1. Total RNA Extraction

RNA was extracted from the rat uterine horn strips and grafted uterinestrip using the RNeasy Fibrous Tissue Mini Kit.

2. Quality Test of RNA with a Spectrophotometer and a Bioanalyzer

Concentration of the extracted RNA was measured with a NanaoDrop. AlsoRIN measurements using a bioanalyzer (6000 nano kits) was carried out. A6000 pico-kit was used for bioanalyzing of the two samples with lowyield (Sample ID: 202, 507).

3. Reverse Transcription Reaction (A) Standard Substance

cDNA obtained by reverse transcription reaction from 1000 ng/uL ofcommercially available Rat Tissue Universal Reference Total RNA (CatalogNumber PR-UR-100, ZYAGEN Inc.) using SuperScript VILO Master Mix(hereinafter “Standard”) was used as a standard substance.

(B) Specimen

Based on the concentration measured with a spectrophotometer, reversetranscription reaction of the specimen with Super Script VILO Master Mixwas performed to give 50 ng/uL of cDNA (the following example). As forthe samples with low yield, that is Sample ID: 202, 502, 507, thereverse transcription reaction was performed to give 25 ng/uL, 25 ng/uL,and 12.5 ng/uL of cDNA, respectively.

4. TaqMan Reaction

TaqMan reaction by using Step One Plus Real-Time PCR System wasperformed with standard 250, 62.5, 15.625, 3.9, 0.98 ng/well or sample5.0 ng/well. A cycle of reaction, two minutes at 50° C. and 10 minutesat 95° C., followed by 15 seconds at 95° C. and one minute at 60° C.,was repeated 40 cycles, then the fluorescence value FAM was measured inreal time after the reaction at 60° C. In addition to the target eightgenes, GAPDH gene as an internal control gene was measured. All thestandards and samples were measured in triplicate.

5. Gene Expression Quantification by Relative Standard Curve Method

From each of the two calibration curves (target gene, GAPDH gene)obtained from the standard, gene expressions level of the target geneand GAPDH gene of the sample was calculated, respectively. Then, thetarget gene expression level was corrected by the GAPDH gene expressionlevel, the internal control gene. And then, an average gene expressionlevel of the samples in each group was calculated, followed by theaverage gene expression level of the two groups was used as a standardto compare with each of the other groups.

[3] Experiment Results Target Gene Expression Analysis Results

TABLE 5 Expression level ng n Era Erb PR Aromatase VEGF IL-6 MCP-1 PEDFSham group 3 55.5 ± 0.7  0.28 ± 0.03 248.5 ± 11.7 — 1.52 ± 0.12 0.71 ±0.16 7.00 ± 1.16 2.77 ± 0.18 E2 control 6 44.2 ± 18.9 2.37 ± 1.07 101.0± 40.3 — 1.62 ± 0.43 14.24 ± 8.74  29.47 ± 20.81 4.51 ± 1.39 SR 0.1mg/kg 6 39.3 ± 14.3 4.05 ± 1.88  94.6 ± 37.7 — 1.44 ± 0.43 9.77 ± 9.7841.95 ± 21.68 6.44 ± 1.56 SR 0.3 mg/kg 6 47.6 ± 14.9 3.05 ± 1.02 176.2 ±81.1 — 1.65 ± 0.40 6.01* ± 3.01  28.30 ± 10.93 6.62* ± 1.84  SR 1 mg/kg6 33.2 ± 20.7 2.08 ± 1.51 127.8 ± 89.1 — 1.33 ± 0.57 6.74 ± 4.96 26.70 ±18.30 5.93 ± 3.01

In comparison with the E2 control group, the effect of SR16234 on theestrogen receptor gene expression of Era/Erb was not observed, and aslight increase trend was observed on the PR. No effect on the geneexpression of VEGF relating to angiogenesis promotion was observed, but,as shown in FIG. 2, an increase trend was observed on the geneexpression of PEDF, that is an angiogenesis inhibitor. Further, as shownin FIG. 3, the gene expression of IL-6, that is an inflammation relatedcytokine, was dose-dependently inhibited. These results on the geneexpression illustrated that the drug had an inhibitory effect oninflammation associated with endometriosis, and angiogenesis.

INDUSTRIAL APPLICABILITY

The estrogen receptor α-inhibiting β partial agonist of the presentinvention can be administered orally with a low toxicity and safeanti-proliferative activity in ectopic endometrial tissues, thus it maybe widely used as therapeutic agents for estrogen-dependentgynecological diseases such as endometriosis, uterine fibroids, anduterus adenomyosis.

1. An estrogen receptor α-inhibiting β partial agonist represented bythe following formula (a);


2. A gynecological disease therapeutic agent comprising the estrogenreceptor α inhibiting β partial agonist of claim 1, a pharmaceuticallyacceptable salt thereof, or a hydrate of the afore-mentioned as anactive ingredient.
 3. The gynecological disease therapeutic agentaccording to claim 2, wherein the gynecological disease comprisesendometriosis, uterine fibroids, and/or uterus adenomyosis.